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Shipboard sampling procedures

Offshore interstitial water sampling using rhizon samplers

Rhizon sampling was undertaken where the structure of the sediments allowed the insertion of rhizon samplers. A standard 3.8 mm diameter drill bit was used to drill a hole in the plastic liner. A spacer on the drill bit prevented the bit from penetrating the core material. Core catcher materials were sampled in a split liner after they were delivered from the drill floor. If necessary, a 2.5 mm wide stainless steel stick was used to prepare a hole in the sediment. Rhizon samplers were placed in a beaker with pure water prior to use. Sample vials were prepared with preservatives (Table T5).

Rhizon samplers were carefully pushed into the sediment and connected to a 50 or 20 mL disposable syringe. A vacuum was established by pulling the syringe plunger and keeping it open with a wooden spacer. The sampling interval was variable and determined by the availability of suitable core material and the amount of drill mud contamination. If the pore water flow through a rhizon sampler was slow, the syringe was taped to the liner to allow core curation procedures to continue. Alternatively, the rhizon samplers were removed and cores were logged and resampled with fresh rhizon samplers after logging.

Where drill mud contamination was negligible, 30 min to 2 h was required for the syringe to fill with pore water. If there was still pore water flow, the syringe was emptied into a 20 mL scintillation vial (Greiner; polypropylene) and reattached. Filtering was generally not necessary because the maximum pore width of the rhizon samplers is 0.2 µm. Broken rhizon samplers can easily be detected, as the vacuum cannot be maintained when the porous tube is damaged. If detected early, damaged rhizon samplers were replaced. Samples collected from broken rhizon samplers were refiltered with 0.45 µm disposable syringe filters (Nalgene 25 mm; nylon). When pore water collection was very slow, two rhizon samplers were used in the same sampling interval to speed up the sampling procedure. Rhizon samplers retain 130 µL of liquid when wet and are prepared for use by being placed in a beaker with pure water. Ideally, the first drops of residual water would have been discarded to avoid collecting it. However, at very low expected sample volumes it was decided to leave this water in the sample and account for the dilution. Approximate sample volumes were measured volumetrically using the syringe grading. Gravimetrical measurement was not possible because of the ship movement.

Effect of drilling procedures on interstitial water quality

Cuttings were flushed from both the HQ and American Petroleum Institute pipes with drill mud composed of guar gum mixed with seawater. Guar gum is a natural, biodegradable polysaccharide that gels in the presence of calcium ions. Guar gum is nonionic and stable in seawater. Upon microbial degradation, the mixture, if undisturbed, becomes acidic (pH ~4).

The ship had a dedicated pump to transfer seawater from the moonpool to the mud tank in order to premix the drill mud. During the drilling process, drill mud flushes the barrel and leaves the pipe through mudways between the barrel shoe and the inner side of the drill bit. Thus, porous or coarse sediments might be expected to contain pore waters mixed with drill mud. Drill mud can clog rhizon samplers, resulting in slow flow rates. This was especially true in the loose sands and disturbed sediments.

Random sampling of seawater from different holes and transects and different drill mud compositions were acquired for reference. Both the guar gum powder and the different substances used on the drill bit and the barrel were sampled as well, in order to provide baseline tracer references for possible contamination. However, because of the filtering effect of the rhizon samplers, the pore water collected was free of contamination.

Seawater sampling

Seawater samples from 2–3 sites along each of the four transects were collected in order to measure oxygen isotopes, carbon isotopes of dissolved inorganic carbon, and uranium concentrations. Samples were collected from the sea surface using a bucket deployed over the side of the vessel, attached to a line. At some sites, they were collected at the same time and from the same side of the vessel as the conductivity, temperature, and density profiling.

For oxygen isotope analyses, crimp glass vials were filled with 20 mL of unfiltered seawater, leaving a small headspace to prevent breakage during shipping. For carbon isotope analysis, 20 mL of unfiltered seawater was placed into a crimp glass vial, and 0.2 mL of mercuric chloride (HgCl2) solution was added. Again, headspace was required to prevent the glass vial from breaking because of temperature changes. For uranium concentration analyses, 2 mL of concentrated ultra pure nitric acid was added to 200 mL of filtered seawater and stored at room temperature.

Sample labeling

Both the syringes and sample vials were hand labeled with hole, core, section, and sampling depth information. All sample information was entered into the OffshoreDIS. The primary sample label was used for the anion 8 mL Nalgene vial. Additionally, all samples and sample splits were labeled with a sequential number in red. This number is useful to quickly label temporary sample containers in the laboratory and for sorting samples. The sequential number was entered into the remarks field of the OffshoreDIS. Samples that were preserved with nitric acid were also marked with a red identifying dot on the lid.

Samples were collected in the curation container and measured for pH, alkalinity, ammonia, and approximate salinity in the geochemistry container. Results were compiled to calculate alkalinities and calibrated ammonia concentrations. These results, the total sample and split-sample volumes, and the type and amount of added preservatives were entered into the OffshoreDIS. Split-sample labels from the OffshoreDIS were glued to the appropriate vials and covered with transparent tape. If label fields failed to print correctly, they were hand corrected with a permanent marker. Samples other than pore water were only hand labeled.

Sample splitting

When possible, water samples split from the primary sample vial were subsampled using a syringe. Where exact amounts were needed (e.g., alkalinity and sulfide), adjustable pipettes (Eppendorf 5000 µL and Eppendorf 1000 µL) were used to transfer samples from the primary sample vial. Samples for isotope analyses were put into vials and sealed without headspace (unlike the strategy for seawater). Table T5 shows the sample-split priority.

The minimum sample amount from which all offshore parameters were measured was ~500 µL. Alkalinity and pH were measured from a 200 µL sample split, and ammonia was measured from a 1:5 dilution (200 µL sample + 800 µL pure water). Salinity can be optionally measured from a ~100 µL sample split. Because rhizon samplers contain 130 µL of pure water when wet, this amount was considered a dilution of the original sample. When the total sample volume collected was low, the remaining sample, after alkalinity titration, was stored in case additional samples were needed. The exact amount of acid added to the alkalinity sample was recorded on the label.

Pure water

Pure water was generated in the curation container from the ship’s tap water with a Seradest USF 3000 purifying cartridge. The conductivity of the water was controlled to be <0.1 µS (>10 MΩcm) by an LFM C1 conductivity detector. Pure water for laboratory use was produced in batches of 10 L and stored in a carboy. For microbiological use, the pure water was additionally treated with an Aquafine ultraviolet disinfection unit to remove any microbial contamination from the cartridge and filtered with a 0.2 µm filter cartridge. The casing of the ultraviolet disinfection system is made up of a quartz tube and stainless steel and, therefore, sterile pure water is potentially not completely metal free. Both pure water and tap water were sampled repeatedly.

Sample temperature

In situ temperatures of the samples could not be measured; however, water temperatures at the sites were generally around 28.5°C, as measured using a YSI 6600V2 conductivity, temperature, and density probe accurate to ±0.15°C. Temperatures in the geochemistry container were well controlled to between 23° and 25°C. For pH measurement, temperature compensation was done using the container temperature, measured with an external temperature sensor. Because measurements were performed in 1.5 or 2 mL Eppendorf vials, temperatures were expected to be equilibrated to ambient temperature. In certain weather situations, however, the air conditioning in the laboratory container failed, causing the ambient temperature to rise well above 25°C. Measurements under such circumstances were avoided, but if they were unavoidable, were noted.

pH value

Sample pH was measured with a Mettler Toledo InLab 423 microcombination glass electrode with a 3 mm tip connected to either a WTW pH 340i pH meter or a Radiometer TIM840 Autotitrator. The pH value and alkalinity were determined from 0.7 mL of sample in 2 mL Eppendorf cups. When the sample volume was smaller, the amount required could be reduced to a minimum of 200 µL using a conical 1.5 mL Eppendorf cup. A constant reading was achieved by turning the vial around the electrode with a special magnetically driven vial holder rather than stirring the sample with a stir bar. The pH meter was calibrated once a day using AppliChem color coded National Bureau of Standards (NBS) scale pH buffer solutions of pH = 4.01 and 7.00. Temperatures were monitored with a pt1000 T-sensor next to the sample vial. The instrument shows the pH with a resolution of 0.001 pH units and the measurement has an accuracy of better than ±0.02 pH units.


Alkalinity was determined by titration with 0.01M HCl where the equivalence point was detected by titrating known amounts of sample (usually 0.7 mL) while measuring the pH value. A Radiometer TIM840 Autotitrator with a 5 mL burette was used for titration and stopped at pH < 3.9. The algorithm used to calculate alkalinity accounts for the activity of seawater and dilution by the titration solution so that the results are comparable for different end-point pH values. The measurement has an accuracy of better than 0.2 mM.

For the titration, a 0.3 mm internal diameter polytetrafluoroethylene (PTFE) tube from the digital burette was placed in the liquid before the titration started. Both the PTFE tube and the pH electrode were rinsed with pure water and carefully dried with laboratory tissues before the measurement. The magnetically driven rotating vial holder is described in more detail in the ESO curation container cookbook. The algorithm is a corrected version of the algorithm of Grasshoff (1983):

ALK [M] = (10–pHinitial/fH+initial) + [(cHCl · VHCl)/V0] –
(10–pHfinal/fH+final) · [(V0 + VHCl)/V0]


  • pHinitial = original pore water pH,
  • pHfinal = pH at end-point of titration (usually pH 3.95),
  • fH+ = activity coefficient of H+ (for standard seawater this is 0.755),
  • cHCl = concentration of titration solution (usually 0.01M),
  • VHCl = titration volume depending on alkalinity of sample, and
  • V0 = initial volume of sample (usually 0.0007 L [700 µL]).


Ammonium was detected using the PTFE tape gas separator technique, as described in the curation container cookbook (modified after Hall and Aller, 1992). With this technique, ammonium is stripped from a 100 µL sample by an alkaline carrier solution (0.2 M sodium citrate in 10 mM NaOH), passes a 200 mm × 5 mm PTFE membrane as ammonia (NH3), and is redissolved as NH4+ in an acidic solution (1 mM HCl). The NH4+ causes a conductivity signal in the acidic carrier that is detected with an Amber Science 1056 conductivity meter with a model 529 temperature-compensated microflow-through cell. The conductivity signal was recorded with a Knauer strip-chart recorder, and peak height was analyzed manually. This method is very precise and stable, practically insensitive to matrix changes, and shows a linear conductivity response for ammonium concentrations between 10 and 1000 µM. The detection limit is 5 µM, and accuracy is <2%.

Generally, measurements were made on the original sample, which was only diluted if the sample volume was extremely low or the ammonium concentration exceeded the calibrated range (>1.6 mM). Ammonia calibration standards were freshly prepared from a 55.6 mM (1000 ppm) standard solution using artificial seawater as a matrix.

A 300–400 µL sample split was taken from the primary sample vial with a Hamilton 1000 µL precision glass syringe and injected onto a 100 µL loop with a Rheodyne high-pressure liquid chromatography valve. The valve was then opened to the carrier solution stream to start the analysis. The Hamilton syringe was rinsed with pure water twice before and after analysis.


Salinity was optionally measured using a Krüss Optronic digital refractometer. The measurement precision for the refractometer is 0.1%, so the data can only be used to detect samples with salinities that differ significantly from seawater. The zero point calibration was performed with pure water. The refractometer was calibrated externally by measuring International Association for the Physical Sciences of the Ocean (IAPSO) standard seawater with a salinity of 34.99.

Headspace sampling

Headspace gas samples were taken and preserved, but methane concentrations were not measured offshore. Ideally, 5 mL sediment was sampled from the working half of the core using a cut-off disposable syringe. The amount was noted in both the laboratory book and in the OffshoreDIS. The sample was immediately transferred into a 20 mL headspace vial containing 10 mL of brine. The receiver solution was composed of a 333 g/L NaCl brine with 24 mg/L HgCl2 to prevent microbial degradation of the sample. The absolute amount of HgCl2 in one vial is 0.24 mg. The vials were crimp sealed, labeled, and stored in a padded box. All gas samples collected were labeled with a red sequential number that matched the sequential numbers of the corresponding pore water samples.

Onshore geochemistry of interstitial water

A total of 118 interstitial water samples (18, 20, 2, and 78 from transects HYD-01C, HYD-02A, RIB-02A, and NOG-01B, respectively) were taken from the cores using rhizon samplers, along with 35 reference water samples (i.e., pure water used onboard for chemical analysis, rain water, tap water, seawater from the drilling site, and filtrate of drilling mud). Alkalinity, pH, salinity, and ammonium were measured from the interstitial water onboard. With the assistance of the technical staff at the University of Bremen, an additional 30 chemical species were measured in the interstitial water. Chloride, bromide, and sulfate were measured by ion chromatography. Na, Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, P, Pb, S, Si, Sr, Te, Ti, V, Zn, and Zr were measured by inductively coupled plasma–optical emission spectrometry (ICP-OES).

Interstitial water analyses

Using analytical equipment housed in the Department of Geosciences, University of Bremen, aliquots of interstitial water samples taken during drilling operations were analyzed for a suite of dissolved species to complement shipboard analyses (Expedition 302 Scientists, 2006).

Cations measured by inductively coupled plasma–optical emission spectrometry

Dissolved cations were measured using a PerkinElmer Optima 3300 R simultaneous ICP-OES. Cation aliquots of pore water samples used for these analyses were acidified directly on board following shipboard sampling with 1% (10 µL acid/1 mL sample) of concentrated HNO3.

During the Onshore Science Party, pore water aliquots were diluted 1:100 for measurements of Na, 1:10 for other major elements (Al, B, Ba, Ca, Fe, K, Li, Mg, Mn, P, S, Si, Sr, and Ti), and 1:40 for trace elements (Al, As, Be, Cd, Co, Cr, Cu, Mn, Mo, Ni, P, Pb, Te, Ti, V, Zn, and Zr) using a 1.0% HNO3 solution (prepared with subboiled distilled HNO3 and 18 MΩcm water). Major and trace elements were analyzed using a cross-flow nebulizer and CETAC USN 5000AT ultrasonic nebulizer, respectively. For measurements of major and trace elements, measuring the intensity at each wavelength was performed in triplicate. Wavelengths used for identifying major and trace elements are shown in Table T6.

In all cases, standardization was done against multielement solutions prepared from commercial standards and adjusted to a NaCl concentration similar to that in the seawater matrix of the samples, as shipboard salinity measurements determined all samples had seawater-like salinities. Calibration standards for major elements were prepared with high-purity single-element standards (Spectrascan by Teknolab, Norway) using a 0.5 M NaCl (Merck certipur) solution as the matrix. Calibration standards for trace element analyses were prepared by multiple dilutions of a multielement standard (Merck Certipur multielement standard IV) prepared with a 0.6 M NaCl solution as the matrix. Measurement precision is ±3% and ±5% for major and trace elements, respectively.

Anions measured by ion chromatography

Unacidified aliquots of the samples were diluted 1:100 using 18 MΩcm water. Concentrations of Cl, Br, and S were measured using 861 Advanced Compact IC with Sequential Compression by Metrohm AG and an IC-Anion column Metrosep A Supp 5 (100 mm run length) at the University of Bremen. Measurement precisions for Cl, Br, and S were ±0.6%, ±3.5%, and ±2% (1σ), respectively. Dilutions of IAPSO seawater served as a certified reference material. Calibration standards were prepared from certified commercial single-anion standards (CertiPUR by MERCK).

Onshore analysis of sediments

X−ray diffraction methods

To determine the mineralogy of the deep forereef slope at Noggin Pass (transect NOG-01B), samples from Hole M0058A were taken every 50 cm for the entire length of the hole. Samples were freeze-dried and ground in agate mortars to ensure sample homogeneity and a fine grain size (<20 µm) so as not to introduce mineral orientation biases during measurement. X-ray diffraction measurement and pattern analyses were performed in the Crystallography research group laboratories (University of Bremen, Central Laboratory for Crystallography and Applied Material Sciences, ZEKAM, Department of Geosciences). X-ray diffraction was measured on a Philips X’Pert Pro multipurpose diffractometer equipped with a Cu-tube (Kαλ 1.541, 45 kV, 40 mA), a fixed divergence slit of 0.2503°, a 16 sample changer, a secondary Ni-filter, and the X’Celerator detector system. Measurements were performed as a continuous scan from 3.0060° to 84.9800°2θ with a step size of 0.0170°2θ (calculated time per step was 50.1650 s). Mineral identification was performed using the Philips software X’Pert HighScore, which gave a semiquantitative value for the weight percent of each identified mineral on the basis of relative intensity ratio (RIR) values. RIR values are calculated as the ratio of the intensity of the most intense reflex of a specific mineral phase to the intensity of the most intense reflex of pure corundum (I/Ic) referring to the “matrix-flushing method” of Chung (1974).

Replicate measurements of three samples suggest that the analytical uncertainty is ±3 wt% for aragonite; ±2 wt% for calcite and Mg calcite; ±5% for quartz; ±5% for total feldspars; and ±7% for total clays. Unfortunately, RIR values are sparse for clay minerals and long chain organic compounds, hampering the quantification in this case. Clay mineral identification and quantification is also hampered in these samples by the low clay weight percent and the tendency of the peak positions of the clay minerals to be sensitive to the chemical composition of the clay minerals (Vogt et al., 2002; Vogt, 2009). Interpretation of clay mineralogy and abundance should therefore be treated with caution and assumed to be of a qualitative nature. The quantification of weight percent Mg calcite is also hampered here by the reference data being limited to only two examples of Mg calcite ([Mg0.10Ca0.90]CO3 and [Mg0.14Ca0.86]CO3). Where the Mg content of Mg calcite deviates from those of the reference data, additional uncertainty will be introduced into the estimation of weight percent for Mg calcite.

Total organic carbon methods

To determine the concentration of total organic carbon (TOC) of the deep forereef slope at Noggin Pass (transect NOG-01B), samples were taken every 50 cm for the entire length of Hole M0058A. Selected sediment samples were analyzed for total carbon and TOC concentrations using a LECO CC-125 carbon-sulfur analyzer at the University of Bremen. Total inorganic carbon was calculated as the difference between total carbon and TOC. Sediment samples were freeze-dried and finely ground by hand in an agate mortar. Approximately 50 mg of dried, ground sample was weighed in a ceramic cup and heated in a furnace. The evolved CO2 was then measured with a nondispersive infrared detector. A second aliquot of 1 g was weighed in a ceramic cup, reacted with 12.5% HCl twice, washed with deionized water twice, and reanalyzed as above. The CO2 measured in the second run was assumed to come from organic carbon. The analytical precision is about ±0.02%.